Licensing Transcription Activator Activity with Ubiquitin Time Clocks

Cells of complex higher organisms rely on information contained in their DNA genomes to provide instructions for development and how to respond to changes in their environment. The majority of information in the cell's genome is stored in genes that can be accessed by reading them, resulting in so-called gene expression. Gene expression is fine-tuned by the action of many different factors, amongst which activator proteins play a pivotal role. Activators exhibit potent activity that recruits all the other necessary factors for the expression of their target genes. They are therefore indispensable for accessing the information contained in genes but at the same time their strong activity poses a serious threat, as excessive gene expression can lead to genome instability and mutation. Accordingly, failure to correctly regulate the activity of activators often has catastrophic pathological consequences for an organism, including cancer cell formation, hormone imbalance, and either autoimmunity or low immune function.

Despite the serious penalties associated with activator dysfunction, little is understood about how cells regulate their activity. Recently, data from our lab and others showed that many potent activators are unstable proteins because they are fused with chains of the small protein ubiquitin, a signal for activator degradation. From these data it has been hypothesized that cells provide activators with a limited license for activity and when this license expires the activator is degraded. While this is an attractive mechanism for keeping activators in check, the molecular identity of this license and the length of its validity remain elusive. Our latest genetic experiments suggest that we are now able to trap activators in their fully licensed states by mutating enzymes that potentially modify the length of ubiquitin chains fused to activators. Here we will investigate these enzymes in detail as they point to ubiquitin chain length being an adjustable time clock for activator activity. Using genetic, genomic, and biochemical tools we will identify all ubiquitin chain modifying enzymes involved in this process and assess their role in licensing activator activity. Unravelling the mechanisms by which activator activity is fine-tuned will provide deep insight into how higher organisms -from humans to plants- regulate access to information contained within their genomes. Such fundamental knowledge offers the foundation for designing new medicines or therapies to tackle a range of human diseases and for modernizing agriculture with novel crop protection strategies.

Gene expression plays a pivotal role in the development of eukaryotic cells and their response to the environment. Failure to precisely program cellular gene expression often has pathological or deleterious consequences. Expression of most genes is initiated by the concerted action of transcription activators and coactivators that exhibit potent intrinsic activities to recruit RNA Polymerase II. While necessary for gene expression, this potent transcriptional activity poses a threat to the cell, as excessive gene transcription may lead to genome instability. Therefore, eukaryotic cells tightly control the deployment of (co)activator activities, but how this is achieved is largely unknown. Surprisingly, degradation of many (co)activators by the ubiquitin-mediated proteasome (UPS) is necessary for the activation of their target genes, indicating that in these cases transcription requires continuous delivery of fresh (co)activator to gene promoters. However, it remains unknown why this seemingly wasteful mechanism is used by the eukaryotic cell. In recent genetic and biochemical experiments we found that knocking out ubiquitin chain elongating E4 enzymes (UBE4) may trap (co)activators in their active states and render transcription of their target genes UPS independent. For the first time these data strongly support a model in which ubiquitination initially boost (co)activator activity before transitioning into a signal for UPS-mediated degradation, thereby averting excessive transcription initiation. To investigate this potential ubiquitin time clock for the licensing of (co)activator activity, we propose to identify and characterize further UBE4s and their counterparts, deubiquitinases (DUB). Using genetic, genomic, and advanced biochemical approaches we aim to map the opposing actions of UBE4s and DUBs to the transcriptome, and provide a direct biochemical link between the remodelling of ubiquitin chain length and transcriptional activity of unstable (co)activators.

The proposed research will have far reaching impacts for a variety of sectors and the wider public. Understanding the mechanisms by which gene transcription is activated in plants is essential to agricultural developments. Human activities increasingly impose constraints on agriculture and threaten future food security. These constraints result in crops having to face a multitude of abiotic and biotic stresses. Resistance or tolerance against these stressors is provided by reprogramming of the transcriptome to prioritize stress defences over normal cellular functions. Consequently, crops either engage in a lengthy and costly battle against disease or succumb under disease pressure, both of which cases results in severely reduced crop yield. Indeed, a reduction in marketable crop yields cost the UK and global economies billions every year and still renders much of agriculture subsidized. A greater understanding of the mechanisms that control specific gene transcription programs to cope with stresses will offer academic scientists and the commercial private sectors novel avenues for exploration of methods to significantly increase crop yields under human-imposed agricultural constraints. Increasing yields is not just desirable but a necessity for a rapidly growing population that demands food security, while avoiding increases in food prices as experienced in recent times. Moreover, our proposed research will offer indications how to directly engineer and improve the plant's existing defence mechanisms. This is a particularly attractive approach, because it meets the sustainable standards that are now appropriately demanded for agriculture by both policy makers and the public. Indeed, timely enhancement of the plant's own defences to optimize crop yields avoids the use of harmful chemicals and pesticides that may pose significant hazards to local biodiversity and even public health.

Our work and that of others show that the fundamental principles that underpin transcription initiation are strongly conserved within the eukaryotic domain. Indeed, a fundamentally similar mechanism of transcription initiation further explored in this proposal is thought to be utilized by pivotal transcription activators in humans, including the estrogen receptor and the oncoproteins c-Myc and SRC-3. This indicates that the impact of our findings will cross over into biomedical research advances, particularly those related to research into different types of cancer and disorders caused by hormone imbalance. The direct beneficiaries of this will be located in both the biomedical academic community and the private commercial pharmaceutical sector, while ultimately the design of new medicines and therapies will improve quality of life and promote healthy aging of the wider public.